Biomedical subjects

Zoltán Szeltner

Publications and source records attributed to Zoltán Szeltner.

10 recordsLinked to original sources

PubMed2006

Kosmotropic salt activation and substrate specificity of poliovirus protease 3C.

Picornaviruses produce a large polyprotein, which is cleaved by virally encoded cysteine peptidases, picornain-2A and -3C. Picornain-3C has characteristics of both the serine peptidase chymotrypsin and the cysteine peptidase papain in that the 3D structure resembles chymotrypsin, but its nucleophile is a cysteine SH rather than a serine OH group. We investigated the specificity of poliovirus picornain-3C (PV3C) protease and the influence of kosmotropic salts on catalytic activity, using FRET peptides related to a cleavable segment of the virus polyprotein. The peptidase activity of PV3C was found to be 100-fold higher in the presence of 1.5 M sodium citrate. This activation was anion-dependent, following the Hofmeister series citrate(3-) > SO4(2-) > HPO4(2-) > acetate- > HCO3(-) > Cl-. The activation appeared to be independent of substrate sequence and arose primarily from an increase in kcat. A shift to higher pH was also observed for the pK1 of the enzyme pH-activity profile. Experiments with the fluorescent probe ANS (1-anilino-8-naphthalene sulfonate) showed that the protease bound the dye in the presence of 1 M sodium citrate but not in its absence or in the presence of 1 M NaCl. Structural changes in PV3C protease were detected using circular dichroism and the thermodynamic data indicated a more organized active site in the presence of sodium citrate. PV3C protease was also activated in D2O, which was added to the activation by citrate. These effects seem to be related to nonspecific interactions between the solvent and the protein. Our data show that the catalytic efficiency of PV3C protease is modulated by the composition of the environment and that this modulation may play a role in the optimal processing of polyprotein for the virus assembly that occurs inside specific vesicles formed in poliovirus-infected cells.

3C Viral Proteases↗

PubMed2006

Properties of the prolyl oligopeptidase homologue from Pyrococcus furiosus.

Prolyl oligopeptidase (POP), the paradigm of a serine peptidase family, hydrolyses peptides, but not proteins. The thermophilic POP from Pyrococcus furiosus (Pfu) appeared to be an exception, since it hydrolysed large proteins. Here we demonstrate that the Pfu POP does not display appreciable activity against azocasein. The autolysis observed earlier was an artefact. We have also found that the pH-rate profile is different from that of the mammalian enzyme and the low pK(a) extracted from the curve represents the ionization of the catalytic histidine. We conclude that some oligopeptidases may be true endopeptidases, cleaving at disordered segments of proteins, but with very low efficacy.

Base Sequence↗

PubMed2005

Crystallization and preliminary crystallographic analysis of porcine acylaminoacyl peptidase.

Acylaminoacyl peptidase (also known as acylamino-acid-releasing enzyme or acylpeptide hydrolase; EC 3.4.19.1) is an unusual member of the prolyl oligopeptidase family catalysing the hydrolysis of an N-acylated peptide to an acylamino acid and a peptide with a free N-terminus. Acylaminoacyl peptidase purified from porcine liver has been crystallized in mother liquor containing 0.1 M Tris-HCl pH 7.0, 10%(w/v) polyethylene glycol 8000, 50 mM MgCl2 and 1%(w/v) CHAPS using the hanging-drop vapour-diffusion technique. A full data set to 3.4 A resolution was collected at ESRF beamline ID14-4 and space group C222 was assigned, with unit-cell parameters a = 84.8, b = 421.1, c = 212.0 A and four molecules in the asymmetric unit.

Animals↗

PubMed2005

Flexibility of prolyl oligopeptidase: molecular dynamics and molecular framework analysis of the potential substrate pathways.

The flexibility of prolyl oligopeptidase has been investigated using molecular dynamics (MD) and molecular framework approaches to delineate the route of the substrate to the active site. The selectivity of the enzyme is mediated by a seven-bladed beta-propeller that in the crystal structure does not indicate the possible passage for the substrate to the catalytic center. Its open topology however, could allow the blades to move apart and let the substrate into the large central cavity. Flexibility analysis of prolyl oligopeptidase structure using the FIRST (Floppy Inclusion and Rigid Substructure Topology) approach and the atomic fluctuations derived from MD simulations demonstrated the rigidity of the propeller domain, which does not permit the substrate to approach the active site through this domain. Instead, a smaller tunnel at the inter-domain region comprising the highly flexible N-terminal segment of the peptidase domain and a facing hydrophilic loop from the propeller (residues 192-205) was identified by cross-correlation analysis and essential dynamics as the only potential pathway for the substrate. The functional importance of the flexible loop has been also verified by kinetic analysis of the enzyme with a split loop. Catalytic effect of engineered disulfide bridges was rationalized by characterizing the concerted motions of the two domains.

Animals↗

PubMed2005

Unclosed beta-propellers display stable structures: implications for substrate access to the active site of prolyl oligopeptidase.

Prolyl oligopeptidase is implicated in the metabolism of neuropeptides and is involved in amnesia and depression. It contains a peptidase and an unusual beta-propeller domain that excludes large peptides and proteins from the active site. The propeller consists of seven blades not closed by a "Velcro" between the first and last blades. The propeller domain was expressed as a stable, soluble protein, P(7). Its conformational identity with that of the native propeller was verified by circular dichroism and digestion with trypsin. Differential scanning calorimetry, kinetic denaturation with urea and equilibrium denaturation with guanidinium chloride have shown that the propeller is more stable than the parent prolyl oligopeptidase. The deletion of the seventh blade of P(7) led to a stable structure, a six-bladed propeller, P(6), which immediately dimerized, in contrast with the monomeric P(7). Addition of an 11 amino acid residue extension to the C terminus of P(6) also produced a dimer, whereas the P(6) labelled with a His-tag at the N terminus displayed a monomer structure. The stability of P(6) and its variants was lower than that of P(7). The denatured propellers refolded readily. This study shows that the unclosed P(7) is a stable structure, and suggests that an opening between the peptidase and the propeller domains is more important for the substrate entry than is the putative opening between the first and seventh blades. Our results suggest that the propellers are simple, versatile structures, which can be prepared artificially.

Anilino Naphthalenesulfonates↗

PubMed2004

His507 of acylaminoacyl peptidase stabilizes the active site conformation, not the catalytic intermediate.

Acylaminoacyl peptidase is a member of the prolyl oligopeptidase family. Amino acid sequence alignment suggests that the stabilization of the tetrahedral intermediate should be mediated by His507 rather than by a tyrosine residue found in the other family members of this serine peptidase group. The pH dependence of k(cat)/K(m) did not reveal any effect of His507. Substitution of an alanine for His507 gave the same bell-shaped pH rate profile with the same pK(a) values (7.0 and 8.7). However, the value of the rate constant was 85 times lower with the modified enzyme, which indicated that His507 is an important residue that is probably involved in the formation of the 3-dimensional structure.

Amino Acid Sequence↗

PubMed2004

Concerted structural changes in the peptidase and the propeller domains of prolyl oligopeptidase are required for substrate binding.

Prolyl oligopeptidase contains a peptidase domain and its catalytic triad is covered by the central tunnel of a seven-bladed beta-propeller. This domain makes the enzyme an oligopeptidase by excluding large structured peptides from the active site. The apparently rigid crystal structure does not explain how the substrate can approach the catalytic groups. Two possibilities of substrate access were investigated: either blades 1 and 7 of the propeller domain move apart, or the peptidase and/or propeller domains move to create an entry site at the domain interface. Engineering disulfide bridges to the expected oscillating structures prevented such movements, which destroyed the catalytic activity and precluded substrate binding. This indicated that concerted movements of the propeller and the peptidase domains are essential for the enzyme action.

Catalysis↗

PubMed2003

Electrostatic environment at the active site of prolyl oligopeptidase is highly influential during substrate binding.

The positive electrostatic environment of the active site of prolyl oligopeptidase was investigated by using substrates with glutamic acid at positions P2, P3, P4, and P5, respectively. The different substrates gave various pH rate profiles. The pKa values extracted from the curves are apparent parameters, presumably affected by the nearby charged residues, and do not reflect the ionization of a simple catalytic histidine as found in the classic serine peptidases like chymotrypsin and subtilisin. The temperature dependence of kcat/Km did not produce linear Arrhenius plots, indicating different changes in the individual rate constants with the increase in temperature. This rendered it possible to calculate these constants, i.e. the formation (k1) and decomposition (k-1) of the enzyme-substrate complex and the acylation constant (k2), as well as the corresponding activation energies. The results have revealed the relationship between the complex Michaelis parameters and the individual rate constants. Structure determination of the enzyme-substrate complexes has shown that the different substrates display a uniform binding mode. None of the glutamic acids interacts with a charged group. We conclude that the specific rate constant is controlled by k1 rather than k2 and that the charged residues from the substrate and the enzyme can markedly affect the formation but not the structure of the enzyme-substrate complexes.

Animals↗

PubMed2002

Substrate-dependent competency of the catalytic triad of prolyl oligopeptidase.

Prolyl oligopeptidase, a serine peptidase unrelated to trypsin and subtilisin, is implicated in memory disorders and is an important target of drug design. The catalytic competence of the Asp(641) residue of the catalytic triad (Ser(554), Asp(641), His(680)) was studied using the D641N and D641A variants of the enzyme. Both variants displayed 3 orders of magnitude reduction in k(cat)/K(m) for benzyloxycarbonyl-Gly-Pro-2-naphthylamide. Using an octapeptide substrate, the decrease was 6 orders of magnitude, whereas with Z-Gly-Pro-4-nitrophenyl ester there was virtually no change in k(cat)/K(m). This indicates that the contribution of Asp(641) is very much dependent on the substrate-leaving group, which was not the case for the classic serine peptidase, trypsin. The rate constant for benzyloxycarbonyl-Gly-Pro-thiobenzylester conformed to this series as demonstrated by a method designed for monitoring the hydrolysis of thiolesters in the presence of thiol groups. Alkylation of His(680) with Z-Gly-Pro-CH(2)Cl was concluded with similar rate constants for wild-type and D641A variant. However, kinetic measurements with Z-Gly-Pro-OH, a product-like inhibitor, indicated that the His(680) is not accessible in the enzyme variants. Crystal structure determination of these mutants revealed subtle perturbations related to the catalytic activity. Many of these observations show differences in the catalysis between trypsin and prolyl oligopeptidase.

Animals↗

PubMed2002

Role of the oxyanion binding site and subsites S1 and S2 in the catalysis of oligopeptidase B, a novel target for antimicrobial chemotherapy.

Oligopeptidase B is a member of a novel serine peptidase family, found in Gram-negative bacteria and trypanosomes. The enzyme is involved in host cell invasion, and thus, it is an important target for drug design. Oligopeptidase B is specific for substrates with a pair of basic residues at positions P1 and P2. The sensitivity of substrates to high ionic strength suggests that the arginines interact with the carboxylate ions of the enzyme. On the basis of a three-dimensional model, two carboxyl dyads (Asp460 and Asp462 and Glu576 and Glu578) can be assigned as binding sites for arginines P1 and P2, respectively. The dyads are involved in several events: (i) substrate binding, (ii) substrate inhibition at high substrate concentrations (different inhibitory mechanisms were demonstrated with substrates bearing one and two arginine residues), (iii) enzyme activation at millimolar CaCl2 concentrations with substrates having one arginine, and (iv) interaction of Ca2+ with the dyads which simplified the complex pH dependence curves. Titration with a product-like inhibitor revealed the pK(a) of the carboxyl group that perturbed the pH-kcat/Km profiles. The OH group of Tyr452 is part of the oxyanion binding site, which stabilizes the transition state of the reaction. Its role studied with the Tyr452Phe variant indicates that (i) the catalytic contribution of the OH group depends on the substrate and (ii) the catalysis is, unusually, an entropy-driven process at physiological temperature. The NH group of the scissile peptide bond accounts for the deviation of the reaction from the Eyring plot above 25 degrees C, and for abolishing potential nonproductive binding.

Anions↗